The present invention relates to a system, reagents, and methods for detecting analytes in fluids. More specifically, but not exclusively, the present invention is directed at electrochemical immunoassay systems, and reagents for detecting and analyzing analytes in fluid samples and methods thereof.
Electrochemical biosensors have been used in in vitro diagnostics for determining the presence and concentration of certain biologically significant analytes in biological samples such as blood, urine, and saliva. Diabetic blood glucose monitoring has been one of the most common and successful commercial applications of electrochemical biosensors. Other diagnostics biosensor applications have been developed and include lactate, cholesterol, creatinine, blood gases, and electrolytes. Both AC and DC electrochemical measurement techniques are employed including amperometry, potentiometry, coulometry, and impedance. The majority of the current biosensor technology relies on selected, free enzymes as a bio-recognition element for the analytes. Further, this technology typically can accurately measure a relatively high concentration of the analytes in the mM range. Consequently, electrochemical detection can be accomplished using a macro electrode without the use of amplification techniques.
Other analytes of interest are found in much lower concentrations compared to glucose. Such analytes include: drugs of abuse, such as, amphetamine, cocaine, phencyclidine (PCP), and tetrahydrocannabinol (THC); therapeutic agents, such as, theophylline, digoxin, digitoxin, and methotrexate; environmental pollutants, such as, PCB and atrazine; biowarfare agents, such as, anthrax, botulism, and sarin; proteins; and hormones.
Various affinity-base assay techniques that use labels have been explored to detect these analytes. The affinity-based techniques include the use of: enzyme labels, radioisotopic labels, chemilluminescent labels, fluorescent labels, and electrochemical redox labels. However, many of these techniques are labor intensive requiring many steps that are best performed in a laboratory by skilled technicians. The number and complexity of steps prohibit routine use of these techniques “in the field”. Many of these tests utilize variations on the competitive Enzyme Linked Immunosorbent Assays (ELISA). Examples include atrazine assays from Strategic Diagnostics and EnviroLogix, Inc. both of which have many manual steps including a 15 minute and 1 hour incubation time respectively. Similar ELISA-based assays and other immunoassay formats will be found that can be applied to a diverse set of assay across many industries but few are capable of a rapid onsite quantitative assay. One of the most commonly available immunoassay formats used for rapid testing or point of care devices is known as lateral flow assays and utilizes immunochromatography. Most of these products are “screening assays” that provide a qualitative result (positive/negative) indicated by the presence or absence of a line. Results are often visually read and often hard to interpret when minor or partial lines are present. Most of these assays require follow-up with another method such as GC/MS or HPLC if the result is positive. There is a great need to provide a technology to these diverse industries to allow rapid affinity-based detection. Fast detection allows rapid actionable results.
The use of electrochemical redox labels, which are also referred to as electron transfer agents or electrochemical mediator labels, have been shown to provide practical and dependable results in affinity-based electrochemical assays. However, the use of electrochemical detection techniques for quantifying the redox labels and, consequently, correlating the concentration of the redox labels with the analyte concentration, has not been without problems. Electrochemical measurements are subject to many influences that affect the accuracy and sensitivity of the measurements, including those related to the proper selection of the mediator conjugate to variations in the electrode structure itself and/or matrix effects derived from variability of the samples.
U.S. Pat. No. 5,589,326 and WO 96/25514 disclose mononuclear osmium complexes comprising two bidentate ligands and one imidazole bound via its ring-nitrogen atoms to the central osmium. These osmium complexes can be used as redox mediators especially in electrochemical biosensors. Nakabayashi et al., Sensors and Actuators B 66 (2000):128-130 examined the evaluation of Os (II) complexes as mediators accessible for biosensors. Mononuclear Os (II) complexes were synthesized and the redox potentials of Os (III/II) complexes could be lowered by the use of 4,4′-dimethyl-2,2′-bipyridine, imidazole and chloride ion as ligands.
US 2003/0096997 describes mononuclear transition metal complexes and there use as redox mediators. As metal atom cobalt, iron, ruthenium, osmium, or vanadium is used. Two bidentate ligands and two other ligands are bound to the central metal. Csoregl et al., Anal. Chem. 1994, 66, 3131-3138 discloses a glucose-sensing layer made by cross-linking glucose oxidase with a polymer derived of poly (vinylimidazole), made by complexing part of the imidazoles to [Os (bipyridine) 2Cl]+/2+.
Many immunoassays require a detection limit much lower than what is currently possible with the electrochemical detection on a conventional macro-electrode. Therefore, signal amplification techniques must be used for these assays to significantly improve the electrochemical detection limit.
In light of the above-described problems, there is a continuing need for advancements in the relevant field, including improved systems, methods, compositions, and reagents related to enhancing the detection analysis of various analytes including therapeutic drugs, drugs of abuse, disease state, analytes for food testing, analytes of environmental importance, and biowarfare agents. The present invention is such an advancement and provides a variety of benefits and advantages.